The overall hypothesis of this proposal is that a detailed characterization of the signal transduction properties of constitutive activated tyrosine kinases associated with human leukemias will identify the critical targets that are required for transformation of hematopoietic cells. These targets can then be exploited for the development of novel therapeutic approaches to leukemia. During the previous funding period, we accomplished each of the goals that were set forward. In particular, we cloned and characterized the transforming properties of a spectrum of tyrosine kinase fusions associated with recurring chromosomal translocations in human leukemias, using both cell culture systems and murine models of leukemia. These include, in addition to the TEL/PDGFbetaR and TEL/ABL fusions, the HIP1/PDGFbetaR, H4/PDGFbetaR, TEL/JAK2 and TEL/TRKC fusion proteins. The availability of a spectrum of constitutively activated tyrosine kinases provides a unique opportunity to identify critically important pathways of transformation through comparative analysis of the signal transduction pathways and target genes activated by these fusion proteins, with validation in animal models. In Specific Aim 1, we will address the hypothesis that characterization of the signal transduction pathways and target genes activated by TEL/PDGFbetaR, TEL/JAK2, TEL/TRKC, and TEL/ABL, and BCR/ABL, will identify critical pathways and targets that are required for transformation. We will utilize standard approaches to analysis of signal transduction, as well as expression arrays. In Specific Aim 2, we will test the hypothesis that the putative critical effectors of transformation identified in Specific Aim 1 can be evaluated and validated in our model systems. We will utilize a combination of approaches that focus on inhibition of transformation in cell culture systems, as well as murine bone marrow transplant models of leukemia using hematopoietic progenitors from donor mice are that are genetically deficient in one or more of the putative targets. In Specific Aim 3, we will test the hypothesis that acute leukemia phenotypes are a collaboration between constitutively activated tyrosine kinases and transcription factor fusion genes such as AML1/ETO, AML1/EVI1 and NUP98/HOXA9. We hypothesize that co-expression of tyrosine kinase fusions and transcription factor fusions will cause an acute leukemia phenotype in murine models, and that these leukemias will retain sensitivity to specific tyrosine kinase inhibitors. This proposal will provide insights into signal transduction and target genes critical for transformation of hematopoietic cells, as well as novel therapies for leukemia.